Interpretation of southern hemisphere humpback whale diet via stable isotopes; implications of tissue-specific analysis

Blubber and skin are commonly used tissues in stable isotope analysis for the purpose of investigating cetacean diet. Critical comparison of tissue-specific isotopic signals is, however, lacking resulting in uncertainty surrounding the representativeness and therefore utility of different tissues for accurate determination of recent foraging. This study used remotely biopsied blubber and skin tissues from southern hemisphere humpback whales for strategic comparison of δ13C and δ15N values. Samples were collected between 2008–2018 as part of long-term monitoring under the Humpback Whale Sentinel Program. Blubber tissues were lipid-extracted prior to analysis, whilst mathematical lipid-correction was performed on skin samples. Isotopic values from paired blubber and skin samples from the same individuals were compared to assess whether tissues could be used interchangeably for isotope analysis and dietary interpretation. Significant differences were observed for both δ13C and δ15N, flagging previously undocumented methodological considerations, and the need for method validation and standardisation in application of these approaches. This study therefore advances methodological aspects of cetacean dietary analysis. This is of elevated importance in the context of rapidly changing ocean ecosystems.

tissue-specific variability in BSI values for the interpretation of diet was further investigated by creating a krill space (isotope range) for each tissue. The tissue-specific krill range would predict the range of which is expected for  13 C and  15 N values to lay within if the individual whale is feeding exclusively on prey item; Antarctic krill (Euphausia superba) as in accordance with the SHHW's classical feeding model. Two sample years, 2013 and 2015 was included in the visualisation as these years showed the greatest variability between the two tissues. Almost all lipid-corrected skin data points fell within the expected krill range of this tissue, whilst less than half of lipid-extracted blubber isotope data fell within the range expected this tissue.
The evaluation and comparison of  13 C and  15 N values in SHHW blubber and skin tissue contribute new methodologically based understanding of bulk stable isotope research. Not only does this research highlight the variation in δ 13 C and δ 15 N values found in lipid-extracted blubber and lipid-corrected skin tissue, but it further underscores the growing importance of emphasizing tissue selection criteria for improved accuracy of the interpretation of long-term dietary results of SHHWs and therefore monitoring of Southern Ocean health.

Subject
Animal Physiology

Specific subject area
North Stradbroke Island, southeast Queensland, Australia Table  Figure Equation

Type of data
How the data were acquired The preparation system used is a Europa EA-GSL interfaced to a SERCON Hydra 20-20 isotope ratio mass-spectrometer (IRMS).

Description of data collection
In situ biopsy sampling from free swimming southern hemisphere humpback whales. A total of 171 paired blubber and skin biopsies were obtained with a modified 0.22 calibre rifle (Paxarms NZ) and flotation darts. Biopsies were immediately sub-sectioned, with the lipid fraction sub-sectioned at blubber core depth 3-4 cm. Blubber and skin samples were stored on ice in the field, and then transferred to -20 °C freezers until bulk stable isotope analysis (BSIA).
NB. Migration direction (south or north) and sex (male or female) was recorded for all sampled whales, however statistically, there was no difference in δ 13 C and δ 15 N values within/ between tissues from either migration direction nor sex, thus these varables were not included in further analysing in this study.
Approximately 30 mg of blubber was lipid extracted prior to BSIA. The solvent lipid extraction of blubber tissue was completed using a modified methanoldichloromethane-water (2:1:0.8 v/v/v MeOH/CH2Cl2/H2O) method.
The mass balance approach for lipid-correction developed by Fry (2002) have been evaluated and suggested to be the best for for lipid correction for SHHW skin tissue according to Groß et al. (2021). Thus this model was applied in our study with the application of isotopic discrimination factor of 8.92 ‰.

Data accessibility
With the article

Value of the data
• The data is useful as it is a long-term data collection of paired blubber and skin tissue samples from 2008-2018 used for bulk stable isotope analysis. • Research involving long-term monitoring of SHHWs dietary trends and the health of the Southern Ocean though bulk stable isotope analysis and fatty acid research. • Critical comparison of tissue-specific isotopic signals is, however, lacking resulting in uncertainty surrounding the representativeness and therefore utility of different tissues for accurate determination of recent foraging. This study therefore advances future methodological aspects of cetacean dietary analysis.   Tables: Table 1: Bulk difference: Table overview of the mean, standard deviation (SD) and range for δ13C and δ15N values for lipid-extracted blubber and lipid-corrected skin tissue of E1 humpback whales (n=171).